ABSTRACT: Pneumocystis pneumonia (PCP) remains a serious complication of AIDS.1-5 Pc thrives during impaired lymphocytic immunity, leading to respiratory failure.3,6-8 To better understand host responses during CD4 deficiency, we studied host innate immunity in PCP, demonstrating central roles for alveolar macrophages (AMS) in Pc clearance.9 We also defined activity of Pc associated inflammation from AMS and alveolar epithelial cells (AECs) leading to respiratory impairment.10,11 Interactions of Pc ?-glucans with the C-Type Lectin Receptor (CLR) Dectin-1 and interactions of Pc MSG with the CLR Mincle initiate clearance of Pc, but also trigger damaging lung inflammation.4,12-16 Understanding mechanisms to restore balance between Pc clearance and inflammation holds potential benefit for treating severe PCP. Since the last submission, we have compelling new data demonstrating central roles across the CLR family during PCP both in CD4 depleted and in immunocompetent hosts using CARD9-/- mice. CARD9, a Caspase Recruitment Domain (CARD) adaptor protein, is the central molecule through which Dectin-1, Mincle, and other CLRs signal.17 Hence, CARD9-/- mice exhibit a deficiency of most CLR immunity. Notably, CD4-deficient CARD9-/- mice had impaired killing of Pc, with markedly greater Pc burdens and concurrently a dramatic reduction of inflammation compared to wild-type mice. Immune competent CARD9-/- mice also had significantly impaired Pc clearance. Despite this phenotype, the molecular events mediated by CARD9 during PCP are unknown. We hypothesize that impairment of CARD9, a central regulator of major CLR function, leads to broad deficiency critical for Pc clearance and inflammatory responses. This will be addressed through three independent but interrelated Aims. In Aim 1, we will evaluate the role of CARD9 in driving Pc clearance and inflammation following interactions with Pc. We will study uptake and killing of Pc, cytokine release, polarization of macrophages, and the cell signaling mechanisms in CARD9-/- versus wild type AMS, AECs and dendritic cells. Next, in Aim 2, we will determine the impact of CARD9 on Pc clearance, inflammation, and mortality in immune competent and CD4 deficient CARD9-/- mice during PCP. We will examine the major causes of lethality during PCP, namely Pc burden and lung inflammation by performing kinetic studies of Pc-infected CARD9-/- mice using both CD4 depleted and immunocompetent mice. Finally, in Aim 3, we will explore cell based treatments targeting CARD9 as adjunctive therapies for PCP. While cell based treatment strategies are not needed for mild to moderate PCP, such therapies may be beneficial in addition to antibiotics in severe PCP with mortality over 80%.5,18 We have demonstrated that transfer of M2 polarized macrophages during PCP signficantly enhances Pc clearance and improves lung inflammation.19 Therefore, we will test the utlility of macrophages driven to overexpress CARD9 and CLR as adjuctive therapy during severe PCP. These studies will define the activity of the important CLR/CARD9 system in host defense during PCP, which may be exploited for novel therapies for Pc infection.